Mycoplasmas, the smallest free-living microorganisms, are widely distributed in nature and commonly produce disease in plants, insects, and animals, including man. Although the economic impact of pathogenesis and effective methods of control are unavailable. The development of mycoplasma genetics has been limited by a lack of genetic tools such as selectable markers (e.g. auxotrophic mutants), methods to transfer DNA (e.g. conjugation, transformation, and transduction), and vectors suitable for cloning. The goal of this proposal is to develop mycoplasma genetic tools, with the long-range goal of using these tools to study the basic biology and disease pathogenesis of these organisms. We have recently established that plasmid pAM120, containing the streptococcal transposon Tn916, can be transformed into Acholeplasma laidlawii, Mycoplasma pulmonis, and Mycoplasma hyorhinis using a polyethylene glycol-mediated procedure. Transformed cells can be selected by their expression of the tetracycline resistance determinant, tetM, located on Tn916, Tetracycline-resistant transformants contain Tn916 inserted into the mycoplasmal chromosome, with different transformants containing Tn916 inserted at different sites; Tn916 apparently behaves as a transposable element in these mycoplasmas. Thus, Tn916 is a potentially important new tool that can be used to study mycoplasma genetics. In addition, we have recently established that the streptococcal replicon in plasmid pVA868 functions in A. laidlawii. Thus, replicons of Gram-positive origin may be useful for the development of mycoplasma vectors, To further develop mycoplasma genetic tools, we propose to: (1) isolate and characterize mutants of M. pulmonis, thereby demonstrating the utility of Tn916 as an insertions mutagen of this organism; (ii) develop a mycoplasma cloning vector by combining broad-host-range Gram-positive replicons with a selectable marker (the tetM gene); and (ii) determine whether the conjugative properties of Tn916 can be used of establish gene transfer techniques in mycoplasmas.